An estimated 1.5 million people die each year from invasive fungal infections, and many millions more are afflicted by debilitating mucosal and subcutaneous mycoses. Current antifungal therapies have serious deficiencies including poor efficacy, limited spectrum of activity, patient toxicity and the emergence of resistant fungi. Consequently, mortality rates have remained disturbingly high. New and improved therapeutic options are desperately needed to improve patient outcomes and redress the rise of resistance. Yet the discovery and development of new pharmocotherapies remains a frustratingly inefficient process. The objective of phase 1 (R21) of this proposal is to apply an unconventional chemical screening strategy to identify physiologically active, and fungal selective inhibitors of fatty acid (FA) biosynthesis. Our approach will focus upon fatty acid synthase (FAS) and the Ole1p FA desaturase, both of which have a fundamentally different structural organization and functional constitution compared to their mammalian counterparts. FAS and Ole1p are both essential for the viability of infectious fungi in vivo, including the prevalent human pathogens Candida albicans and Cryptococcus neoformans. We propose to exploit these targets to develop a new class of efficacious and broad spectrum antifungal therapy. A new whole-cell based approach developed within our lab termed Target Abundance based Fitness Screening (TAFiS), will be applied to identify specific inhibitors of C. albicans FAS and Ole1p. This method facilitates the selection of chemical probes that interact with a specific target protein within intact cells, thereby combining the advantages of traditional target- and cell- based screens into a single high-throughput assay. Inhibition of FA synthesis will be confirmed through biochemical analysis of treated fungal and mammalian cells, and those with fungal selective activity identified. In phase 2 (R33), the antifungal potency, selectivity and ADME properties of lead compounds will be optimized, and structure-activity relationships established. The spectrum of activity of selected leads will also be tested against important human fungal pathogens, and to isolates resistant to current antifungal drugs. Finally, the biopharmaceutic, pharmacokinetic and toxicologic properties of selected leads will be assessed before antifungal efficacy is tested in a mouse model of disseminated fungal infection. Completion of this study will facilitate the development of a new generation of antifungal drugs that can cure invasive fungal infections that are refractory to current treatment options.